1. Field of the Invention
The present invention relates to the expression of human insulin in P. pastoris and, more particularly the invention is related to the field of DNA recombinant technology and to the production of insulin precursors in host microorganisms such as yeast. More precisely, the invention refers to a recombinant methylotrophic yeast strain for producing human insulin precursors. The invention also relates to DNA constructions and methods for obtaining the strains. The inventive strain comprises, in its genome, at least one copy of a first DNA construction and one copy of a second DNA construction, wherein said constructions are capable of expressing and secreting an insulin precursor.
2. Description of the Prior Art
It is well known that diabetes is usually treated with insulin injections, e.g. of human insulin. Insulin is an essential hormone in metabolism and is a protein consisting of two polypeptide chains, namely A chain and B chain. A chain comprises 21 amino acid residues and B chain comprises 30 amino acid residues, and both chains are covalently connected by disulfide bridges in the positions A7–B7 and A20–B19, and by an intra-chain disulfide bond connecting residues A6–A11.
Insulin is produced in the pancreas by the β cells of the Langerhans islets as preproinsulin. Preproinsulin consists of a prepropeptide having 24 amino acids acting as an export signal sequence followed by a peptide named proinsulin and containing 86 amino acid residues. Said preproinsulin may be represented by: prepeptide-B-C-A, wherein the C peptide is a connecting peptide comprising 31 amino acid residues and chains A and B are chains A and B of proinsulin.
When the preproinsulin chain is synthesized, the signal peptide directs synthesis towards the endoplasmic reticulum of the β cells and at that moment the signal peptide splits out, secreting the proinsulin into the endoplasmic reticulum.
Then, during packing of the insulin molecule within the secreting system of the β cells, the C peptide is cleaved, thus liberating the native insulin molecule, which is appropriately “folded”. Cleavage of the C peptide is carried out through the action of enzymes acting upon the proinsulin dibasic sequences.
Presently it is known that the C peptide has an important function in the formation of the tertiary structure of the insulin molecule.
The production of insulin for treating diabetes has been a concern in the pharmaceutical industry for many years. Since the development of recombinant DNA techniques, a wide variety of methods for the production of insulin in microorganisms have been published.
Bacteria were the first host microorganisms employed in recombinant DNA techniques, particularly Escherichia coli (E. coli). In the first experiments using E. coli, strategies similar to those used in the production of synthetic insulin were employed. According to these methods, chains A and B were cloned and expressed independently in the host microorganisms, thus obtaining two polypeptides corresponding to chains A and B. The native insulin was then obtained by performing the steps of forming disulfide bonds between chains A and B and the respective intra-chain bridge in vitro. This oxidizing process was carried out as described by Chance, R. E. et al., in Diabetes Care 4:147; 1981; and Goedel, D. V. et al. in Proc. Natl. Acad. Sci. U.S.A. 76: 106–110, 1979. One of the biggest difficulties presented by this method was the random formation of disulfide bonds, which generated molecules with an incorrect tertiary structure. Through this method, the yield of native insulin with biological activity was extremely low, thus increasing production costs dramatically.
In view of the above difficulties, experts have introduced the idea of cloning the DNA sequence of proinsulin or its derivatives, where the C peptide is represented by fragments of different sizes. This idea is based on the fact that the presence of the C peptide or its derivatives produces a higher yield of correctly folded proinsulin after the oxidizing step as compared to the yield resulting from oxidizing chains A and B separately (Dteiner, D. F. et al. Proc. Acad. Sci. 60:622; 1968). Thus, it was observed that chain C which acts as a connecting peptide of chains B and A, allows for cysteine residues to be spatially favored for a correct oxidation. It was demonstrated that the proinsulin molecule formed in this manner could function as a precursor from which insulin could be obtained by in vitro removal of peptide C, using specific enzymes. (Kemmler, W. et al. J. Biol. Chem. 91: 246:6786; 1971). It was also demonstrated that if fragment C of these precursors was changed for a connecting peptide of a smaller size that maintains in vitro cleavable sites at both ends, and, if proper enzyme action was used, equivalent, and in some cases better results were obtained in the production of insulin. These precursors were named mini-proinsulins (Wollmer, A. et al. Hoppe-Seyler's, Z. Physiol. Chem. 355:1471–1476; 1974 and EPO Patent 195 691).
European Patent No. EP 0055945 discloses a process for producing and expressing proinsulin in E. coli and methods for producing human insulin. The production of proinsulin in E. Coli on a large or commercial scale is disclosed in U.S. Pat. No. 5,460,954. U.S. Pat. No. 4,431,740 discloses a DNA having a sequence encoding proinsulin, and another DNA encoding pre-proinsulin, and a microorganism such as E. coli transformed with such sequences.
However, the expression of heterologous proteins in E. coli, has a number of difficulties well known by those skilled in the art. Briefly, the following can be mentioned:
When an E. coli or any prokaryotic microorganism is used as a host for the expression of proteins from eukaryotes, the microorganism is incapable of forming the disulfide bonds which allow for the correct formation of a tertiary structure. As a consequence, when proteins such as human insulin are cloned and expressed in microorganisms, said proteins tend to aggregate forming inactive complexes or inclusion bodies.
Solubilization and purification of proinsulin from the inclusion bodies requires a large number of additional steps. One of these steps comprises dissolving the aggregates with reagents such as urea or guanidine chloride. Subsequently, it is necessary to submit the insulin precursor to an oxidizing agent by oxidative sulfitolysis, wherein the cysteine molecules of both chains adopt the SSO−3 form. Subsequently, the S-sulfonated groups are converted into sulphydryl groups (—SH—) in the presence of a thiolated agent (di-thiotreitol or 2-mercaptoethanol). Finally, these groups are oxidized in presence of oxygen for the formation of the sulfide bonds.
New methods for the recovery of proinsulin from the inclusion bodies are still the aim of several investigations, attempting to improve the yield and achieve a correct folding of the protein which is dramatically reduced by purification of the protein and causes the purification process to be extremely complex. (Chance, R. et al. Proceedings of the Seventh American Peptide Chemistry Symposium, pages 721–728; 1981; Pierce Chemical Company, Rockford, Ill.; Chan, S. J. et al. Proc. Natl. Acad. Sci. USA 78 (9): 5401–5405, 1981, and Frank, B. H. et al. Proceedings of the Seventh American Peptide Chemistry Symposium, pages 729–739; 1981; Pierce Chemical Company, Rockford, Ill.).
In addition, in E. coli or any other prokaryote organisms protein translation starts with a methionine residue. In order to remove the methionine from the amino terminal end the gene of interest is usually cloned as a fusion protein. The removal of insulin from the fusion peptide requires an additional step involving digestion of the peptide with specific proteases. Otherwise, the methionine residue must be removed with cyanogen bromide (CNBr).
European Patent No. 0 055 945 discloses a method and a vector to cleave a proinsulin analog having a smaller C peptide and wherein the methionine residue is removed by treatment with CNBr.
Other difficulties and drawbacks that may be found in the expression of heterologous proteins in prokaryotes is the decrease or reduction of protein stability under the action of cytoplasmic protease. U.S. Pat. No. 5,460,954 discloses a process for producing human proinsulin in E. coli which comprises a vector containing a sequence at the 5′ end of the proinsulin gene, encoding an amino acid sequence which prevents degradation by protease within the cell.
Many investigators are attempting to improve the methods for producing human insulin in E. coli through a simpler method and with better results. These methods for improving protein yield consist in replacing the C peptide by smaller sequences (Chang, Seung-Gu at al. Biochem. J. 329; 631–635, 1998).
Methods for expressing proinsulin in bacteria have also been developed, which combine different procedures such as the expression of a fusion protein comprising a polyhistidine tail in the N-terminal end, a methionine residue and the proprotein sequence of of human insulin, all included in an expression vector for bacteria (Cowley, Darrin J. et al. FEBS Letters, 402: 124–130, 1997).
By reason of the operative drawbacks and difficulties found in the expression of human insulin in prokaryotic hosts, many attempts have been made to obtain high levels of expression of human insulin in eukaryotic hosts such as yeast. Consequently, yeast has become one of the selected hosts for the expression of eukaryotic proteins. These microorganisms provide clear advantages as compared to bacteria in relation to the production of mammalian proteins. Yeast has secretion mechanisms that are similar to those of mammals and has the capacity of properly folding, proteolitically processing, glycosilating and secretingmammalian proteins.
When appropriate vectors are employed in the yeast for exporting the protein outside the cell, the process for recovering and purifying the proteins exported into the culture medium is simpler and has a better yield than the expression in cell cytoplasm. In addition, the secretion system provides an appropriate environment for the formation of the di-sulfide bonds that are necessary for protein folding (Smith, et al. 1985; Science 229:1219). On the other hand, cytoplasm is a reducing environment in which these bonds are not formed. Under these circumstances, production of any proteins requiring di-sulfide bonds for maintaining a correct tertiary structure, as is the case of insulin, will have better results when said proteins are secreted.
A yeast system used as host for the production of a large number of proteins is, for example, the yeast species Saccharomyces cerevisiae. The genetic structure of this yeast has been studied in detail by a number of investigation groups.
Several polypeptides such as insulin have been cloned and expressed in Saccharomyces cerevisiae. The expression of this propeptide may follow the secretory path or may be accumulated in the cytoplasm of the host microorganism. In the event of the accumulation, time consuming and complex purification processes must be employed, the processes requiring steps for the formation of di-sulfide bonds, as disclosed in European Patent No. 37255. In order to avoid these drawbacks and complicated steps, the proinsulin gene sequence is cloned subsequently to an additional DNA sequence named “leader” or signal peptide that originates the pre-proinsulin peptide. This peptide, once recognized and processed by the yeast, provides the secretion of proinsulin into the culture medium.
In addition to the foregoing, any precursors of the proinsulin type that are produced in Saccharomyces cerevisiae undergo a rapid enzymatic process either when expressed in the cytoplasm or when secreted into the medium. It has been demonstrated that human proinsulin is especially sensitive to enzymatic cuts in two dibasic sequences (Arg31–Arg32 and Lys64–Arg65). This causes the cleavage of the molecule before the formation of the di-sulfide bonds, thus resulting in the separate generation of peptides C, A and B.
It has been found that if, instead of proinsulin, shorter sequences are employed wherein the C peptide has been removed or, it is simply represented by shorter fragments having up to two amino acids of lysine, arginine type, a more stable molecule is obtained, which is not digestible by proteases, and capable of been processed in vitro to give a biologically active insulin molecule (Lars Thim et al. Proc. Natl. Acad. Sci. USA 83: 6766–67770; 1986).
European Patent No. 195 691 discloses several precursors, inter alia, those of type B-X-Y-A where B and A correspond to the B and A chains of human insulin, and where X and Y are represented by the amino acids lysine and arginine, these amino acids being digestible by the trypsine and carboxypeptidase B enzymes for their conversion into human insulin. However, while considerable amounts of A0Arg-desB(30) are produced as digestion sub-products, these sub-products do not have amino acid 30 of the B chain while an arginine residue remains connected to the A chain. The arginine residue can not be easily removed and this causes serious inconveniencies in the process of purifying the protein, also considerably diminishing product yields. Total yield of this precursor in Saccharomyces cerevisiae is remarkably low.
On the other hand, U.S. Pat. No. 4,916,212 discloses a simple chain proinsulin precursor, where said precursor is represented by the formula: B(1-29)—(Xn—Y)m—A(1-21), Xn is a peptidic chain of n amino acids, Y is lysine or arginine, n is an integer from 0 to 35, m is 0 or 1, B(1-29) is a B chain lacking the threonine at position 30, and A(1-21) is the A chain of human insulin. This US patent reveals that —Xn—Y— does not have two adjacent basic amino acids, such as lysine and arginine, because the digestion with trypsin produces byproducts that are difficult to separate during the purification steps. The products obtained from these genetic designs do not contain threonine at position 30 and, therefore, they must be subjected to an additional step consisting in the addition of this amino acid by the catalytic action of trypsin in the presence of Thr-Obu ester, as disclosed in U.S. Pat. No. 4,343,898 and Rose, K. et al. Biochem. J. 211:671–676, 1983.
In any case, in addition to all the modifications introduced into the insulin precursors, the expression of these peptides in Saccharomyces cerevisiae has resulted in low yields and scaling drawbacks in heterologous protein production. These problems are generally associated to low efficiency promoters and to the fact that the sequences of interest are cloned in autonomous replication plasmids. These plasmids do not remain uniformly distributed within the culture medium and they usually decrease as the number of copies increases. As a result of this, and after some duplication cycles, cells with 2, 3 or 0 copies of the plasmid used as vector are found in the culture (Chan, S. J. et al. Proc. Natl. Acad. Sci. USA 78 (9): 5401–5405. 1981).
An expression system in yeast, but not using Saccharomyces as a host, is the methylotrophic yeast system. These microorganisms may be very useful as hosts for the expression of heterologous proteins required to be produced in large volumes. Heterologous proteins that are expressed in methylotrophic yeast may be secreted with expression levels that are equivalent to those of E. coli and higher than those of Saccharomyces cerevisiae. 
Mthylotrophic yeasts are unicellular microorganisms capable of growing in the presence of methanol as the only carbon source. This yeast can be kept without trouble in high cellular densities when grown in a high volume fermentor. In addition, this yeast is capable of producing many of the post-translated modifications undergone by the higher eukaryotic cells, such as proteolytic digestions, protein folding, di-sulfide bonds formation and glycosilation.
Pichia pastoris is one of the twelve species within the four yeast genera capable of metabolizing methanol as the only one carbon source (Cregg, J. M. et al. Bio/Technology 11:905–910, 1993). The remaining genera are represented by Candida, Hansenula and Torulopsis. 
These yeasts share a large number of enzymes corresponding to the metabolic pathways of methanol (Veenhuis, M. et al. Adv. Microb. Physiol. 24:1–82, 1983). The first step of this metabolic pathway is the oxidation of methanol into formaldehyde, generating hydrogen peroxide by action of the alcohol oxidase enzyme (AOX).
The cell avoids hydrogen peroxide toxicity by carrying out this first metabolic reaction of methanol in a special organelle named peroxisome.
There are two genes in P. pastoris which encode alcohol oxidase enzymes I and II: AOX1 and AOX2 genes. The AOX2 gene is responsible of most alcohol oxidase activity in the cell (Cregg, J. M. et al. Mol. Cell. Biol. 9:1316–1323, 1989).
The expression of this gene is highly regulated and it is induced by methanol, with AOX1 representing a value close to 30% of the total soluble cell proteins. It is for this reason that the expression systems usually employed with Pichia pastoris include in their vectors the AOX1 gene promoter.
Thomas Kjeldsen et al. compared the expression of proinsulin and precursor peptides of (B1-29-Ala-Ala-Lys-A1-21) insulin in S. cereviciae and in P. pastoris. The products were secreted into the culture medium with the aid of an amino acid sequence that is fused to the leader (amino) end of the precursor. Several signal peptides were employed for determining the secretion efficiency of the insulin precursor such as the pre-pro-peptide αmating factor of Saccharomyces cerevisiae and synthetic derivatives thereof. These pre pro peptides have amino acid sequences useful as targets for the activity of specific proteases resulting in the release of the peptide into the culture medium. All the insulin peptides employed by these authors are secreted into the medium as a precursor lacking threonine at position 30 of the B chain. This product, recovered and purified from the culture medium, had to be submitted to an exhaustive process called transpeptidation. Transpeptidation consists in the addition of threonine and it is disclosed in U.S. Pat. No. 4,916,212 to Markussen et al. It adds a step to the purification process of the insulin molecule.
In the above described state of the art, it has been a concern of the inventors to find a solution to all of the above mentioned problems and drawbacks in the prior art.